Virological Sampling of Inaccessible Wildlife with Drones

viruses Communication Virological Sampling of Inaccessible Wildlife with Drones Jemma L. Geoghegan 1,*,† ID , Vanessa Pirotta 1,† ID , Erin Harvey 2,†, Alastair Smith 3, Jan P. Buchmann 2, Martin Ostrowski 4, John-Sebastian Eden 2,5 ID , Robert Harcourt 1 and Edward C. Holmes 2 ID 1 Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia; [email protected] (V.P.); [email protected] (R.H.) 2 Marie Bashir Institute for Infectious Diseases and Biosecurity, Charles Perkins Centre, School of Life and Environmental Sciences and Sydney Medical School, The University of Sydney, Sydney, NSW 2006, Australia; [email protected] (E.H.); [email protected] (J.P.B.); [email protected] (J.-S.E.); [email protected] (E.C.H.) 3 Heliguy Scientific Pty Ltd., Sydney, NSW 2204, Australia; [email protected] 4 Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, Australia; [email protected] 5 Westmead Institute for Medical Research, Centre for Virus Research, Westmead, NSW 2145, Australia * Correspondence: [email protected]; Tel.: +61-2-9850-8204 † The authors contributed equally to this paper. Received: 12 May 2018; Accepted: 31 May 2018; Published: 2 June 2018 Abstract: There is growing interest in characterizing the viromes of diverse mammalian species, particularly in the context of disease emergence. However, little is known about virome diversity in aquatic mammals, in part due to difficulties in sampling. We characterized the virome of the exhaled breath (or blow) of the Eastern Australian humpback whale (Megaptera novaeangliae). To achieve an unbiased survey of virome diversity, a meta-transcriptomic analysis was performed on 19 pooled whale blow samples collected via a purpose-built Unmanned Aerial Vehicle (UAV, or drone) approximately 3 km off the coast of Sydney, Australia during the 2017 winter annual northward migration from Antarctica to northern Australia. To our knowledge, this is the first time that UAVs have been used to sample viruses. Despite the relatively small number of animals surveyed in this initial study, we identified six novel virus species from five viral families. This work demonstrates the potential of UAVs in studies of virus disease, diversity, and evolution. Keywords: whale; virome; drone; mammalian host; virosphere There is a growing interest in understanding the diversity, evolution, and disease associations of viruses in natural populations [1]. Although sampling of many terrestrial species is relatively straightforward, there may be serious logistical challenges for animals that live in inaccessible habitats. Marine environments are one such habitat [2–4]. It has recently been shown that wild populations can be sampled using Unmanned Aerial Vehicles (UAVs) [5,6]. UAVs are rapidly transforming wildlife science, allowing sampling from dangerous and inaccessible environments to address questions previously only approached by theory. Here, we show how UAVs can be used to sample viruses. This approach may ultimately enable a better understanding of the patterns and drivers of disease emergence in wild populations. There is evidence that marine mammal health is deteriorating as anthropogenic stressors on the world’s oceans increase [7]. However, contemporary assessments of marine mammal health are strongly biased towards animals whose health is already compromised, such as stranded animals, Viruses 2018, 10, 300; doi:10.3390/v10060300 www.mdpi.com/journal/viruses Viruses 2018, 10, 300 2 of 7 which in part reflects the difficulties in sampling aquatic environments. Sampling from free-ranging marine mammals is therefore critical to assess whether healthy animal populations are potential reservoirs of viruses and other transmittable agents. Following the use of UAV technology for sampling, we employed a meta-transcriptomic approach [8,9] to help characterize the virome of an important marine mammal, the Eastern Australian humpback whale (Megaptera novaeangliae), which serves as a model for work in this area. Recent analyses of whale breath, or “blow”, have revealed an extraordinary diversity and abundance of microbiota. Importantly, the microbial communities observed were divergent from those present in the surrounding seawater such that they could be considered as distinctly whale blow associated [5,6]. To date, however, these studies have not included virus sampling, and little is known about the diversity of the whale virome and whether this differs fundamentally from that seen in terrestrial mammals. We collected whale blow samples from 19 humpbacks during the 2017 annual northward migration from Antarctica to northern Australia (Figure1a). To adhere to all Australian legislative requirements, our UAVs were registered with the Civil Aviation Safety Authority (CASA) and operated by a CASA-certified remote pilot. All flights were conducted in good weather (no rain, Beaufort < 3), from a small research vessel, where the UAV was launched and landed on a launch pad at the stern of the boat. A closed, sterile petri dish was placed on eight suction cups on the UAV before each flight. Members of the team visually scanned the area for humpback whales. Once an individual or pod was chosen, the vessel was driven at a constant speed and distance from the whale. Once the respiratory rhythm was determined (i.e., downtime length), the UAV was launched to coincide with surfacing. The UAV pilot was directed by spotters on the vessel and positioned the UAV with the aid of the live feed from a forward-facing camera. To minimize sample contamination, the petri dish remained closed until immediately before the whale surfaced. The dish was remotely opened as the UAV accelerated towards and through the densest part of the whale blow, collecting the maximum amount of sample in the dish and lid (see Video S1). The petri dish was immediately closed and the UAV was returned to the vessel. The petri dish containing the sample was removed from the UAV and secured with Parafilm®. All samples were stored immediately in a portable −80 ◦C freezer. A different whale was sampled each flight. Different individuals within a pod were chosen based upon unique distinctive markings (e.g., white flanks and barnacle arrangements). RNA was extracted using an RNeasy Plus Universal mini kit (Qiagen, Australia). Due to low RNA concentration, all 19 samples were pooled and concentrated using a NucleoSpin RNA Clean-up XS kit (Macherey-Nagel, Australia). A single library was produced for RNA sequencing using the Low-Input SMARTer Stranded Total RNA Sample Prep Kit with Mammalian rRNA depletion (Clontech, Australia), with 1 ng of the pooled whale blow RNA as input. Paired-end (100 bp) sequencing of the RNA library was performed on the HiSeq 2500 platform (Illumina, Australia) at the Australian Genome Research Facility. RNA sequencing of the rRNA-depleted library resulted in 19,389,378 paired reads (100 nt in length) that were assembled de novo into 107,681 contigs. Sequencing reads were first quality trimmed then assembled using Trinity [10]. The assembled transcriptome was annotated based on similarity searches against the NCBI nucleotide (nt) and non-redundant protein (nr) databases using BLASTn [11] and Diamond (BLASTX) [12], respectively, and an e-value threshold of 1 × 10−5. Transcript abundance was estimated using RSEM [13] implemented within Trinity. Our transcriptome data revealed that the humpback whale blow contains a wide diversity of DNA and RNA viruses (that we refer to “whale-blow-associated” viruses). BLAST analysis revealed the relative abundance of taxonomic classes present in the non-rRNA transcriptome data, of which bacteria occupied ~45%, while ciliates were the second-most abundant source at ~29%. Importantly, Baleen whale species contributed 0.9% of the transcriptome data and were the most abundant source of mammalian RNA, indicating our sample is indeed whale associated. Viruses occupied ~0.01% of the non-rRNA transcriptome, which falls within the range of other meta-transcriptome studies of Viruses 2018, 10, 300 3 of 7 Viruses 2018, 10, x FOR PEER REVIEW 3 of 7 bacteriophagevertebratess included [9]. Despite the this S relativelyiphoviridae low abundance,(18.4% of theall viralviruses) contigs and observed the Myoviridae fell into 42 classified (15.2% of all viruses).viral Among families, the including most abundant 29 families viral of bacteriophage families that (Figure are known1b). The to most infect relatively eukaryotes abundant were small single-bacteriophagesstranded (ss) includedDNA viruses, the Siphoviridae specifically(18.4% the of all Circoviridae viruses) and (and the Myoviridae Circoviridae(15.2%-like of virus all viruses).es) (6.5% of all viruses),Among as the well most as abundant members viral of familiesthe Parvoviridae that are known (2.4%) to infect and eukaryotesan RNA virus were smallfamily, single-stranded the Tombusviridae (ss) DNA viruses, specifically the Circoviridae (and Circoviridae-like viruses) (6.5% of all viruses), as well (0.9%).as members of the Parvoviridae (2.4%) and an RNA virus family, the Tombusviridae (0.9%). FigureFigure 1. (a) Map 1. (a) Mapshowing showing the thehumpback humpback whale whale sampling location location (red (red star), star), approximately approximately 3 km off 3 km off the coast of Sydney, New South Wales, Australia. Purple arrows indicate the typical seasonal migratory the coast of Sydney, New South Wales, Australia. Purple arrows indicate the typical seasonal routes of

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